Ethanol causes widespread and varied actions on the central nervous system that are manifested in alterations in normal neurochemical processes and behavior. The cellular and molecular sites of action of ethanol in the CNS have been the focus of intense research over the past 10 years. Many different neuronal processes have been shown to be sensitive to acute and chronic exposure to ethanol. Of these, the large family of neurotransmitter-gated ion channels has been shown to particularly sensitive to ethanol. It can be hypothesized that many of the actions of ethanol on CNS function and behavior are due to selective effects of ethanol on these ion channel proteins. Research in my laboratory and that of others has shown that a subtype of glutamate receptor, the N-methyl-D-aspartate receptor, is inhibited by ethanol at concentrations at are behaviorally relevant During the course of the current funding period, my laboratory has focused on determining the molecular determinants that confer ethanol sensitivity upon these receptors. Using both native and recombinant NMDA receptors expressed in brain neurons and heterologous expression systems, we have shown that the ethanol inhibition of NMDA receptors is modulate both by subunit composition and intracellular processes that interact with the C-termini of NMDA receptor subunits. Our most recent data show that there are calcium-sensitive and insensitive components to the ethanol sensitivity of NMDA receptors. The calcium-sensitive form involves the C-terminus of the NR1 subunit which possesses distinct binding sites for protein phosphorylation and protein-protein interactions. These processes regulate the activity of the NMDA receptor and appear to be selectively altered by ethanol. The calcium-insensitive component of ethanol's inhibition of NMDA receptors is hypothesized to be mediated via an interaction of ethanol with key residues that make up the transmembrane domains of the receptor. In this continuation of a currently funded R01, we propose to precisely define the molecular sites and mechanisms of action that underlie the calcium-sensitive and insensitive forms of ethanol inhibition of NMDA receptors. This research will utilize two- electrode voltage clamp, single-cell calcium imaging and whole-cell patch clamp to measure the effects of ethanol on NMDA receptors expressed in oocytes, HEK 293 cells, and cultured neurons. Both molecular and pharmacological approaches will be used to unravel the complex interplay between receptors and intracellular signaling processes that ultimately determine the ethanol sensitivity of NMDA receptors.